Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
Hemophilia A (HA) is an X-linked bleeding disease resulting from a functional FVIII deficiency, affecting 1:5000 males worldwide. For several decades the HA dog model has been the most extensively used for preclinical studies (1). Notably, in two strains of dogs the underlying mutation consists of an inversion in intron 22 of the FVIII gene that is analogous to the most common human mutation (2). This model faithfully replicates the human disease at both genotypic and phenotypic levels (3,4). To date there is no characterization of the cFVIII protein due to difficulties in purifying large amounts from canine plasma and to the relative poor performance in recombinant FVIII expression systems in general. Although the cFVIII cDNA sequence has a high sequence identity to human FVIII (hFVIII) (5), adult HA dogs develop immune responses upon exposure to hFVIII that preclude the assessment of the efficacy and safety of potential novel therapies for HA. Notably, among humans even small nucleotide changes in the hFVIII gene may predispose to inhibitor formation (6).
Identifying hFVIII variants that exhibit superior coagulation properties are highly desirable. It is an object of the invention to provide such proteins for use as therapeutics.